The present invention relates to an apparatus and method for manufacturing uniform, bilayer vesicles, or liposomes, using either the batch/flow or continuous flow processes.
Liposomes are phospholipid bilayers surrounding an aqueous compartment. Their dimensions range from 0.20 microns to over ten microns in diameter. They may comprise a single bilayer of lipid, a so-called unilamellar vesicle, or they may contain multiple bilayers interspaced by an aqueous phase, or so-called multilamellar vesicles. Liposomes are uniquely suited as carriers for drugs, biological molecules such as DNA, proteins and non-biological molecules such as carboxyfluorescein. These materials can be encapsulated in the interior aqueous spaces of the liposome or they may be inserted in or attached to the lipid bilayers which bound and limit the internal aqueous space. Unilamellar vesicles are often preferred to multilamellar vesicles because of their size, biocompatibility and defined internal space.
Commonly utilized manufacturing procedures for the preparation of liposomes include the simple dispersion of dried phospholipids in an aqueous media using a homogenizer. This procedure results in the formation of multilamellar liposomes. These multilamellar liposomes may be converted to unilamellar liposomes by ultrasonic irradiation or by passage through filters under pressure. Lipids in organic solutions can be injected into an aqueous media. This often results in the formation of unilamellar vesicles.
Many biological molecules to be entrapped are not compatible with organic solvents. Accordingly, another procedure for liposome formation is the detergent batch dialysis method. This method incorporates the co-micellization of lipids with a detergent having a high critical micelle concentration. A micelle is an optically transparent species which is soluble in water. The material to be incorporated is added simultaneously and usually remains in bulk aqueous phase if it is hydrophillic but incorporates into the mixed micelle if it is hydrophobic. The mixture is placed into a suitable dialysis vessel, one boundary of which is defined by a dialysis membrane, and the detergent is removed by dialysis. As the detergent is removed, the lipids, which have a very low critical micelle concentration, remain behind and thermodynamically rearrange themselves into unilamellar vesicles. The detergent batch dialysis procedure is relatively mild with the detergent of choice being typically octyl-D-glucoside which is biocompatible. Typically the batch dialysis method places a volume of the liposome precursor solution in a dialysis bag which forms the vessel alluded to above. The dialysis bag is itself immersed in a volume of a dialysis buffer solution. The entire volume of the buffer is periodically changed.
Several disadvantages are encountered with either of the commonly practiced techniques for producing liposomes. These disadvantages include vesicle rupture due to mechanical stress, size inhomogeneity, excessive time for vesicle formation, limited internal volume of liposomes, inconsistent encapsulation of material, liposomes which may be unstable, dilution of reagents, and temperature effects causing a general lack of reproducibility.
Two commercial instruments are presently available which attempt to circumvent some of the above-listed problems. These two instruments are the LIPOPREP produced by Dianorm, Diachema AG, Rushilkon, Switzerland, and the MICROFLUIDIZER produced by Microfluids Development Corporation, a division of Biotechnology Development Corporation, Newton, Mass.
The first instrument is a batch/flow dialysis unit which removes detergent from detergent/phospholipid mixed micelle solution by a continuous flow os the buffer across two membranes between which is disposed a stationary volume of the mixed micelle solution. The instrument allows for variable flow rates for dialysis and provides a temperature control unit which can be added. The liposomes are prepared in the batch dialysis mode and can produce up to six milliliters of liposomes in up to twenty-four hours. The disadvantage of this instrument is perceived to be the long dialysis time for a small volume of liposomes produced.
The second instrument effects a continuous manufacture of liposomes and microemulsions. The basic device includes a reservoir containing multilamellar vesicles previously prepared by homogenization or by other means and a high pressure unit which propels the multilamellar vesicles through filters for sizing. The filters are changeable as desired. A volume greater than fifteen milliliters can be produced within minutes. The disadvantages of this instrument are perceived to be the lack of temperature control resulting in inherent heating of the equipment during usage, large dead spaces unavailable for dialysis, variance in liposome homogeneity concerning size and internal volumes, and liposome stability. Moreover, the high pressures used can shear the sensitive vesicle damaging molecules such as DNA and other biopolymers. The manufacturing procedure using this instrument is a two-step method which first requires the formation of multilamellar vesicles.
Of the two manufacturing procedures, as represented by the above instruments currently in use, that based on the detergent batch/flow dialysis is perceived as being the most reproducible, is the most mild in the sense that it maintains molecular activity and is the method by which detergent can be most effectively removed. However, it has not been possible yet to accurately define the condition for maximum encapsulation of reagents, high reproducibility, minimization of dialysis time and requisite detergent levels due to lack of data on the kinetics for liposome formation. Thus, consistent manufacturability of liposomes for commercial purposes is not maximized.
In view of the foregoing, it is believed advantageous to provide an apparatus and method for liposome manufacture which allows for real-time kinetic data acquisition of liposome formation. The apparatus and method is believed most desirably practiced by the incorporation of an optical monitoring system which measures light transmission level of the liposomes forming within the liposome precursor solution. Thus, the system and method can be used to prepare and to follow the kinetics of liposome formation whether using a batch/flow or a continuous flow manufacturing process. Moreover, the use of a servo-loop which controls selected manufacturing parameters such as flow rate and/or temperature in accordance with the output of the optical monitoring system can advantageously improve liposome formation and encapsulation performance.